Transcriptional regulation of pyruvate kinase and phosphoenolpyruvate carboxykinase in the adductor muscle of the oyster Crassostrea gigas during prolonged hypoxia.

The response of Crassostrea gigas to prolonged hypoxia was investigated for the first time by analyzing the metabolic branch point formed by pyruvate kinase (PK) and hosphoenolpyruvate carboxykinase (PEPCK). PK and PEPCK cDNAs were cloned and sequenced. The main functional domains of the PK sequence, such as the binding sites for ADP/ATP and phosphoenolpyruvate (PEP), were identified whereas the PEPCK sequence showed the specific domain to bind PEP in addition to the kinase-1 and kinase-2 motifs to bind guanosine triphosphate (GTP) and Mg(2+), specific for all PEPCKs. A C-terminal extension was detected for the first time in eukaryota PK. Separation of mitochondrial and cytosolic fraction showed that more than 92% of the PEPCK enzyme activity was cytosolic in gills, digestive gland, mantle and muscle. PK and PEPCK mRNAs and enzyme activities have been measured in muscle during prolonged hypoxia for 20 days. Adaptation of PK in hypoxic muscle at transcriptional level occurred lately by decreasing significantly the PK mRNA level at day 20 while PK enzyme activity was inhibited by the high content of alanine. The PEPCK mRNA ratio in hypoxic muscle significantly increased at day 10 simultaneously to the PEPCK enzyme activity. Succinate accumulation observed at day 10 and day 20 confirmed the anaerobic pathway of muscle metabolism in oyster subjected to hypoxia. Regulation of C. gigas PEPCK in muscle occurred at gene transcription level while PK was first regulated at enzyme level with alanine as allosteric inhibitor, and then at molecular level under a fast effect of hypoxia.